The long-term objective of this proposal is to develop new technologies for cloning full-length cDNAs of mammalian species. Development of complete sets of full-length cDNA clones and sequences that represent all genes of human and model organisms would be enormously useful for gene discovery and functional analysis. However, current technologies for obtaining full- length cDNA clones are very laborious and time consuming. Comprehensive approaches to the full-length cDNA library construction and full-length cDNA cloning are proposed in this application. The strategy is constructing the full-length first- strand cDNAs that contain an oligo (dT) linker printer at 5' ends and an anchor sequence at 3' ends. The ligation anchored first- strand cDNAs (LA-cDNA) thus made can serve as reusable sources for multiple usage. Specifically, we propose 1) to construct ligation anchored first strand cDNA, 2) to develop a library-free system for cloning mRNA species that are more difficult to be cloned using other methods, 3) to develop a more reliable RACE method for cloning both 5' and 3' cDNA ends together, 4) to construct full-length cDNA libraries using PCR amplified LA-cDNA, and 5) to construct subtractive full-length cDNA libraries in which high abundance sequences are specifically removed. Preliminary results demonstrate that the new approaches are more efficient, and have the potential for large-scale full-length cDNA cloning project for mammalian species.
| Gao, Zhi; Xu, Zhidong; Hung, Ming-Szu et al. (2009) Procaine and procainamide inhibit the Wnt canonical pathway by promoter demethylation of WIF-1 in lung cancer cells. Oncol Rep 22:1479-84 |
